HUMAN MUTATION 22:92^97 (2003)
METHODS
Epigenetic Detection of Human Chromosome 14
Uniparental Disomy
S.K. Murphy,1* A.A. Wylie,1,2 K.J. Coveler,3 P.D. Cotter,4 P.R. Papenhausen,5 V.R. Sutton,3
L.G. Shaffer,3 and R.L. Jirtle1
1
Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina; 2AstraZeneca Pharmaceuticals, Ltd., Alderley
Edge, Cheshire, UK; 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas; 4Division of Medical
Genetics, Children’s Hospital Oakland, Oakland, California; 5Department of Cytogenetics, LabCorp Inc., Research Triangle Park, North Carolina
Communicated by Haig H. Kazazian
The recent demonstration of genomic imprinting of DLK1 and MEG3 on human chromosome 14q32 indicates
that these genes might contribute to the discordant phenotypes associated with uniparental disomy (UPD) of
chromosome 14. Regulation of imprinted expression of DLK1 and MEG3 involves a differentially methylated
region (DMR) that encompasses the MEG3 promoter. We exploited the normal differential methylation of the
DLK1/MEG3 region to develop a rapid diagnostic PCR assay based upon an individual’s epigenetic profile. We
used methylation-specific multiplex PCR in a retrospective analysis to amplify divergent lengths of the
methylated and unmethylated MEG3 DMR in a single reaction and accurately identified normal, maternal
UPD14, and paternal UPD14 in bisulfite converted DNA samples. This approach, which is based solely on
differential epigenetic profiles, may be generally applicable for rapidly and economically screening for other
imprinting defects associated with uniparental disomy, determining loss of heterozygosity of imprinted tumor
suppressor genes, and identifying gene-specific hypermethylation events associated with neoplastic progression.
Hum Mutat 22:92–97, 2003. r 2003 Wiley-Liss, Inc.
KEY WORDS:
uniparental disomy; monochromosomal hybrid; methylation; methylation-specific PCR; DLK1; MEG3;
GTL2; imprinted gene; bisulfite sequencing
DATABASES:
DLK1 – OMIM 176290; GDB: 9958854, GenBank: NM_003836;
MEG3 – OMIM 605636; GDB: 11500849; GenBank: AB032607, AF052114, AK057522, AK055725, AF090934,
AF119863, BC036882
INTRODUCTION
Uniparental disomy (UPD) of chromosome 14 causes
multiple congenital anomalies and has been most
commonly identified in individuals having two maternally derived copies [upd(14)mat] [Sutton and Shaffer,
2000; Kurosawa et al., 2002]. These individuals have
certain phenotypic characteristics, including intrauterine
growth retardation, short stature, scoliosis, hypotonia,
obesity, distinctive facial appearance, mental delay,
developmental delay, and precocious puberty. Paternal
UPD14 [upd(14)pat] is less common and affected
persons have a more severe phenotype as compared to
upd(14)mat, including marked developmental delay,
growth retardation, hirsute forehead, short palpebral
fissures/blepharophimosis, small/abnormal ears, protruding/long philtrum, depressed nasal bridge, short neck,
small thorax, abdominal muscle hypoplasia, joint contractures, and mental retardation [Sutton and Shaffer,
2000; Kamnasaran, 2001; Eggermann et al., 2002].
The phenotypes associated with maternal and paternal
UPD14 have implied the presence of genes subject to
genomic imprinting on chromosome 14. Such genes are
r2003 WILEY-LISS, INC.
characterized by expression from a single parental allele,
unlike the vast majority of genes throughout the genome
that are biallelically expressed. The monoallelic expression of imprinted genes suggests that gene copy number
is an important factor in regulating gene function.
Deleterious phenotypes linked to maternal and paternal
UPD14 may therefore result from either the complete
loss of expression or overexpression of imprinted genes
on this chromosome. Consistent with this idea, mice
having UPD for distal chromosome 12, syntenic to
human 14q32, exhibit reciprocal phenotypes. Paternal
duplication of this region leads to promotion of growth
Received 26 November 2002; accepted revised manuscript 14
March 2003.
*Correspondence to: Susan K. Murphy, Ph.D., Box 3433,
Duke University Medical Center, Durham, NC 27710.
E-mail: murphy@radonc.duke.edu
Grant sponsor: AstraZeneca Pharmaceuticals, Ltd.; Grant sponsor:
NIH; Grant numbers: F32CA94668; K23HD40843; R03HD38433;
R01CA25951; R01ES08823.
DOI 10.1002/humu.10237
Published online in Wiley InterScience (www.interscience.wiley.com).
EPIGENETIC DETECTION OF UPD14
and late embryonal or neonatal lethality while maternal
duplication is characterized by growth retardation and
late embryonal lethality (see MEG3; MIM# 605636; also
see Imprinting Maps of the Mouse, Mammalian Genetic
Unit, Harwell, UK www.mgu.har.mrc.ac.uk/imprinting/
imprin-viewmaps.html).
We have recently established the presence of two
imprinted genes on human chromosome 14 [Wylie et al.,
2000]. DLK1 (delta, drosophila, homolog-like 1; MIM#
176290) and MEG3 (maternally expressed gene 3; also
referred to as GTL2, for gene trap locus 2; MIM#
605636) are paternally and maternally expressed genes,
respectively. They are located approximately 90 kb apart
on human 14q32, a region frequently affected in reported
human UPD14 cases [Sutton and Shaffer, 2000; Coveler
et al., 2002]. DLK1 encodes a 45–60 Kd protein member
of the delta-notch family of proteins [Laborda et al.,
1993; Smas and Sul, 1993] involved in cellular signaling
and differentiation [Laborda, 2000], and may function in
the mitogen activated protein kinase (MAPK) pathway
[Ruiz-Hidalgo et al., 2002]. Evidence of an essential role
for DLK1 in the etiology of upd(14)mat syndrome comes
from a report of Dlk1 null mice that recapitulate
phenotypes associated with upd(14)mat and indicate
that mouse Dlk1 is involved in early embryonic
development, postnatal growth, and fat deposition
[Moon et al., 2002]. The function of MEG3 is currently
not understood. MEG3 is postulated to produce a noncoding RNA transcript due to lack of an extended open
reading frame within the multiple alternatively spliced
mRNAs [Schuster-Gossler et al., 1998].
The identification of paternally and maternally
expressed genes on chromosome 14 along with the
clinical description of UPD14 and the identification of
cytogenetic subgroups with upd risk association [Papenhausen et al., 1999; Berend et al., 2000; Robinson, 2000;
McGowan et al., 2002] prompted us to develop a direct
diagnostic tool for detection of UPD14 based strictly on
differences in the methylation profile of individuals with
UPD14 vs. those with biparental chromosome 14
inheritance.
MATERIALS AND METHODS
Bisul¢te DNA Modi¢cation
Sodium bisulfite modification of DNA was performed based on
the method described by Grunau et al. [2001]. Briefly, 1 mg of
genomic DNA was denatured with 3M NaOH for 20 min at 421C
followed by deamination in saturated sodium bisulfite/10mM
hydroquinone (Sigma; St. Louis, MO) solution, pH 5.0 for 4 hr at
551C. The DNA was desalted using the Wizard DNA Clean-up
System (Promega; Madison, WI), then desulfonated in 3M NaOH
(20 min at 371C) and ethanol precipitated. The samples were
resuspended in 25 ml Tris-Cl, pH 8.0 and stored at 41C.
Methylation-Speci¢c PCR
Bisulfite-treated genomic DNA was subjected to an optimized
methylation-specific PCR protocol in 25 ml reactions using B5 ng
template (B50 ng of non-bisulfite treated DNA where applicable), 3 mM MgCl2, 0.2 mM dNTPs, 0.4 mM each M primer (MF:
GTT AGT AAT CGG GTT TGT CGG C; and MR: AAT CAT
AAC TCC GAA CAC CCG CG) and/or 0.8 mM each U primer
93
(UF: GAG GAT GGT TAG TTA TTG GGG T; and UR: CCA
CCA TAA CCA ACA CCC TAT AAT CAC A) [Kubota et al.,
1997; Zeschnigk et al., 1997a]. The use of desalted primers
(without further purification) gave unexpected results such that a
weak band corresponding to the opposite parental region was
sometimes present in addition to the correct band. In contrast,
primers purified by polyacrylamide gel electrophoresis (SigmaGenosys; The Woodlands, TX) accurately and reproducibly
amplified the anticipated regions and were used for all experiments
shown herein. Touchdown PCR was used as follows: 941C for 3
min followed by five cycles of 941C for 30 sec, 701C for 30 sec,
721C for 30 sec; five cycles of 941C for 30 sec, 651C for 30 sec,
721C for 30 sec; 30 cycles of 941C for 30 sec, 601C for 30 sec, 721C
for 30 sec; final 5 min extension at 721C. The products were
separated on 3% high resolution agarose gels and visualized by
ethidium bromide staining.
Bisul¢te Sequencing
Bisulfite-treated genomic DNA was amplified by PCR (3 min at
941C, then 40 cycles of 941C for 30 sec, 551C for 30 sec, 721C for
30 sec with a 5 min extension at 721C) with primers specific for
the bisulfite converted MEG3 DMR (SQF1: GAT TTT TTT TAT
ATA TTG TGT TTG and SQR: CTC ATT TCT CTA AAA ATA
ATT AAC C) in 25 ml reactions with 5–25 ng template, 3 mM
MgCl2, 0.2 mM dNTPs and 0.4 mM each primer. The 235-bp PCR
products were resolved on an agarose gel, purified using Sigma
GenElute spin columns (Sigma; St. Louis, MO), and cycle
sequenced (Thermo Sequenase Radiolabeled Terminator Cycle
Sequencing Kit; Amersham Biosciences, Piscataway, NJ) using a
nested primer (SQF2: GTG TTT GAA TTT ATT TTG TTT
GG): 951C for 30 sec, 551C for 30 sec, 721C for 1 min for a total of
35 cycles.
RESULTS
In our initial analysis of the epigenetic profile of the
DLK1/MEG3 region we demonstrated that the region
encompassing the MEG3 promoter is differentially
methylated, although we had not determined the
parental origin of the methylated chromosome [Wylie
et al., 2000]. Studies in mice show that like H19
[Kerjean et al., 2000], the paternal chromosome is
methylated at the Meg3 DMR [Takada et al., 2000;
Paulsen et al., 2001]. The extent of the DMR of human
MEG3 was recently shown to span approximately 4 kb of
genomic sequence that encompasses the putative
promoter region and includes two consensus CTCF
(CCCTC binding factor) binding sites that also exhibit
differential methylation [Wylie et al., 2000] (Fig. 1). To
determine the parental origin of methylation at this
DMR as well as the feasibility of analyzing the
methylation status of this region with a PCR-based
method, we utilized DNA from individuals previously
demonstrated to have UPD for chromosome 14 and
DNA from normal human fetal liver tissue. DNA
samples treated with sodium bisulfite [Grunau et al.,
2001] were analyzed by methylation-specific multiplexed
(MSM) PCR. Two independent primer sets were
designed to amplify the methylated and unmethylated
DMR such that the amplicons are distinguished by size
on a non-denaturing agarose gel. As shown in Figure 2,
the primers specific for methylated DNA (M) produced a
160-bp band in the normal and upd(14)pat samples
(lanes 1 and 9), while the primers designed to amplify
94
MURPHY ET AL.
FIGURE 1. Schematic representation of the DLK1/MEG3 locus at
human chromosome 14q32.The paternally expressed DLK1 and
maternally expressed MEG3 are separated by approximately 90
kb.The promoter of MEG3 is situated within an B4-kb di¡erentially methylated region (DMR) containing two consensus CTCF
binding sites (ovals). The region analyzed by MSM-PCR spans
B1,700 bp.The positions of individual CpGs are denoted by lollipops, and the primers used for MSM-PCR and bisul¢te sequencing are represented by arrows. MF and MR, forward and reverse
primers speci¢c to bisul¢te converted methylated DNA; UF and
UR, forward and reverse primers speci¢c to bisul¢te converted
unmethylated DNA; SQF1 and SQR, primers used to generate
PCR amplicons from bisul¢te converted DNA for sequencing
with primer SQF2. Position ‘‘0’’ corresponds to nt 64,450 of
BAC AL117190 and the complement of nt 440,243 of contig
NT _030824.
unmethylated DNA (U) produced a 120-bp band in the
normal and upd(14)mat samples (lanes 2 and 6). When
the primers were multiplexed in the same reactions (M +
U), they again amplified only their respective targets
(lanes 3, 7, and 11). The specificity of the primers for
bisulfite-treated DNA is demonstrated by the lack of
PCR products for untreated DNA (lanes 4, 8, and 12).
These results indicate that the M and U primer sets
specifically amplify the methylated and unmethylated
sequences, respectively, and furthermore that the
maternal chromosome is unmethylated while the paternal chromosome is methylated within this MEG3 DMR.
To carry out a more extensive test of the multiplexed
PCR reaction, we analyzed additional DNA samples from
patients previously diagnosed with UPD14 [Pentao et al.,
FIGURE 2. Methylation-speci¢c PCR primers speci¢cally amplify
the methylated and unmethylated copies of the MEG3 DMR. Bisul¢te-treated (+) or untreated (^) genomic DNA was subjected
to methylation-speci¢c PCR using the M or U primer pairs separately or multiplexed to generate 160-bp and/or 120-bp bands
only from bisul¢te-modi¢ed methylated (lanes 1, 3, 9, and 11)
and unmethylated (lanes 2, 3, 6, and 7) template DNAs, respectively. These bands were absent with untreated genomic DNA
template (lanes 4, 8, and 12). Samples: normal, liver DNA from
a 122d human conceptus; upd(14)mat and upd(14)pat, DNA from
individuals with maternal and paternal uniparental disomy of
chromosome 14, respectively. Lane 13: negative control.
FIGURE 3. MSM-PCR analysis of normal and UPD14 samples.
PCR ampli¢cation of bisul¢te-treated DNA from three individuals with biparental chromosome 14 inheritance (lanes 1^3),
three upd(14)mat (lanes 4^6) and three upd(14)pat (lanes 7^9)
was performed with the U and M primers in multiplexed reactions. Lanes 10 and 11: DNA from monochromosomal hybrid
(MCH) cell lines carrying human maternal or paternal chromosome 14, respectively.The 160-bp and 120-bp amplicons are derived from methylated and unmethylated template DNA,
respectively. Lane E, empty; lane C, negative control.
1992; Papenhausen et al., 1995; Walter et al., 1996;
Cotter et al., 1997; Berend et al., 2000; Towner et al.,
2001a, b; Coveler et al., 2002; McGowan et al., 2002].
The chromosome 14 status of the samples was initially
kept concealed to insure an unbiased assignment by the
MSM-PCR technique. Normal controls included parental DNA samples from two of the UPD patients in
addition to normal human fetal liver DNA. DNA from
three normal, three upd(14)mat, and three upd(14)pat
individuals was treated with sodium bisulfite and
analyzed using MSM-PCR. The predicted UPD status
of each sample based on the MSM-PCR result was
confirmed when the identity of the samples was revealed.
Figure 3 shows representative data obtained for these
DNA samples, with amplification of both the 160- and
120-bp bands for samples with biparental chromosome
14 inheritance (lanes 1–3), the 120-bp band for
upd(14)mat samples (lanes 4–6), and the 160-bp band
for upd(14)pat samples (lanes 7–9). To further corroborate these data, DNA from monochromosomal somatic
cell hybrid cell lines were assessed for methylation status
of the MEG3 DMR. The two monochromosomal hybrid
cell lines were generated from lymphocyte DNA derived
from the same individual such that the maternal and
paternal chromosome 14 were separated into different
rodent–human hybrids [Coveler et al., 2002]. These two
samples also yielded the anticipated PCR products (lanes
10 and 11), based on the known parental identity from
previous marker analysis (data not shown). Together,
these results indicate that the multiplexed PCR reactions
accurately identified the parental origins of chromosome
14 in the UPD samples based on the epigenetic profile of
the MEG3 DMR.
To confirm unambiguously that the identity of the
UPD samples was assigned correctly by the MSM-PCR
assay, we performed bisulfite nucleotide sequencing. The
primers used for PCR amplification and sequencing of
the DMR were designed to anneal to sequence devoid of
CpG dinucleotides. As shown in Figure 4, the methylation profile of each sample was consistent with and
corroborated the results obtained by MSM-PCR. Bisulfite
sequencing of the monochromosomal hybrids showed
that the paternally derived chromosome 14 was
methylated completely while the maternally derived
EPIGENETIC DETECTION OF UPD14
95
FIGURE 4. Bisul¢te sequencing of the MEG3 DMR. Bisul¢te-treated genomic DNA was ampli¢ed by PCR using primers SQF1and SQR
followed by nucleotide sequencing using primer SQF2. Lane order: G A T C. Cytosines in the context of 5 0 -CpG-3 0 in the original
unconverted sequence are designated by arrowheads at the right of panel 11. Normal individuals (N; panels 1^3) display an approximate 50:50 distribution of unmethylated to methylated cytosines. Individuals with upd(14)mat and the somatic cell hybrid carrying
the maternal chromosome14 (M; panels 4^6 and 10) lack methylated cytosines while individuals with upd(14)pat and the somatic cell
hybrid carrying the paternal chromosome 14 (P; panels 7^9 and 11) have methylated cytosines.The panel numbers correspond to the
same sample DNAs analyzed in Figure 3.
chromosome 14 was unmethylated, indicating that the
fusion event did not affect methylation status of the
human chromosome 14 retained in the hybrid cell line.
The three normal samples exhibited an approximate
50:50 distribution of methylated to unmethylated CpGs,
the upd(14)mat samples were unmethylated, and the
upd(14)pat samples were methylated at these positions.
The reproducible detection of faint cytosine bands in one
maternal UPD sample (panel 6, Fig. 4) is indicative of
the presence of a low level (10–25% as quantitated by
Phospho Imager analysis; Molecular Dynamics Storm
860, Amersham Biosciences, Piscataway, NJ) of sitespecific methylation in this sample for two of six CpG
sites analyzed. The conversion of unmethylated cytosines
to uracils (resulting in thymines in the PCR products) by
the sodium bisulfite appeared to be complete (Fig. 4 and
data not shown). However, we cannot rule out the
possibility that certain CpG dinucleotides are refractory
to bisulfite conversion. Previous analyses did not detect
mosaicism for UPD in this region [Pentao et al., 1992].
In addition, mixing experiments showed that the MSMPCR assay accurately detects each parental chromosome
when present at a 10-fold lower concentration relative to
the other parental copy (data not shown), also supporting that mosaicism is an unlikely explanation in this case.
The presence of specific sites of CpG methylation in a
background of unmethylated CpGs is not unexpected.
Previous reports in which individual cloned PCR
products generated from DMRs of bisulfite-treated
DNA have shown low level variability in the parental
methylation status [Zeschnigk et al., 1997b; Vu et al.,
2000; Li et al., 2002]. Importantly, the pattern of
methylation observed in this sample (panel 6, Fig. 4) did
not impede the accurate diagnostic and reproducible
amplification by MSM-PCR (lane 6, Fig. 3).
DISCUSSION
With the recent identification of the imprinted genes
DLK1 and MEG3 on human chromosome 14 and their
association with a differentially methylated region [Wylie
et al., 2000], an unresolved issue remained regarding the
identity of the methylated parental chromosome. By
analysis of documented cases of UPD14, we have shown
here that it is the paternal chromosome that is
methylated in this region, consistent with the demonstration of paternal methylation for the mouse Dlk1/
Meg3 DMR [Takada et al., 2000; Paulsen et al., 2001].
Paternal methylation is an unusual characteristic in
terms of imprint regulation [Reik and Walter, 2001] and
is only thus far known to be shared in humans with the
IGF2/H19 imprinted region on chromosome 11 [Kerjean
et al., 2000].
Using this newly established methylation profile of the
DLK1/MEG3 imprinted domain, we have developed an
assay for detection of UPD14 that eliminates the
requirement for parental DNA and informative microsatellite marker analysis, and reduces the time requirement and expense of traditional diagnostic procedures.
Indeed, the need for analysis of DNA only from the
proband for UPD determination could lead to cost
savings of two-thirds as compared to performing studies
which include parental DNA samples. Further, the
procedure can be completed within one day. It has
96
MURPHY ET AL.
recently been suggested that UPD14 patients may be
going unrecognized in part because many are karyotypically normal [Kurosawa et al., 2002]. The ability to
rapidly screen for UPD14 using MSM-PCR will facilitate
diagnosis of these individuals. Already, this assay has
been successfully used to exclude UPD14 as a major
etiologic component in 200 individuals that exhibit
Prader-Willi like symptoms yet test negative for chromosome 15 abnormalities [Dietz et al., 2003].
One potential limitation of this technique is that
individuals with mosaicism for UPD14 could theoretically be misclassified as having a normally methylated
MEG3 DMR depending on the extent of mosaicism.
Although this was not seen in the assays we performed,
prudent use of further testing will be required for these
individuals if they exhibit phenotypic characteristics
consistent with UPD14.
The utility of this type of assay is not limited to the
detection of uniparental disomies. It can also be
exploited to assess the methylation status of any genomic
region that exhibits differences in methylation status due
to imprinting defects, loss of heterozygosity for imprinted
tumor suppressor genes, or hypermethylation in cancer,
with care taken to reduce potential contamination from
adjacent normal cells.
ACKNOWLEDGMENTS
We thank Robert Waterland, Kay Nolan, and Jennifer
Weidman for critical reading of the manuscript. We also
thank the NIH-supported Laboratory of Human Embryology at the University of Washington for the fetal tissue
used in these studies. This work was supported by grants
from NIH grants F32CA94668 (S.K.M.), K23HD40843
(V.R.S.), R03HD38433 (L.G.S.), and R01CA25951 and
R01ES08823 (R.L.J.), and by funding from AstraZeneca
Pharmaceuticals, Ltd. (A.A.W.). Further information on
genomic imprinting can be found at www.geneimprint.com.
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